The present invention relates to a method for producing proteins by recombinant DNA technology and to permanently transfected host cells for use in the method.
In recent years, recombinant DNA technology has advanced to the stage where, in general, it is readily possible to prepare a gene, that is a DNA sequence which encodes a desired protein. The gene may encode only a desired protein product or may encode a pro- or prepro-protein which, after translation, is cleaved to produce the desired final product. The gene may be prepared, for instance, (1) by isolating messenger RNA (mRNA) and using this as a template for the production of complementary DNA (cDNA) by reverse transcription, (2) by isolating the natural gene from genomic DNA using appropriate probes and restriction enzymes, (3) by synthesizing the gene from its component nucleotides or (4) by using a combination of these techniques.
It is also well known that a prepared gene can be placed in a vector, such as a plasmid or phage vector, under the control of appropriate 5xe2x80x2 and 3xe2x80x2 flanking sequences which allow the gene to be transcribed into mRNA and then translated into protein. Many important 5xe2x80x2 and 3xe2x80x2 flanking sequences, such as translation start and stop codons, TATA boxes, promoters, enhancers and polyadenylation sites, have been identified. The part of a vector including the gene and the 5xe2x80x2 and 3xe2x80x2 sequences is herein referred to as a transcription unit.
In general, the skilled person will be able readily to construct an expression vector and use it to transfect or transform a host cell such that the host cell is able to produce the desired final protein product. A variety of host cells can be used. Early work was carried out using prokaryotic microorganisms such as E. Coli. These hosts had the disadvantages that they do not have the necessary mechanisms to cleave efficiently pro- or prepro- sequences from proteins, they are unable to glycosylate proteins, they cannot cope with genes containing introns, and they generally do not secrete the proteins when formed.
There has therefore been a tendency towards the use of eukaryotic host cells. These generally avoid most of the disadvantages of prokaryotic host cells. However, eukaryotic host cells generally have more stringent requirements for culturing and also have slower growth rates. It is therefore not readily possible to produce large quantities of a product merely by culturing a eukaryotic host cell transformed with an expression vector.
There has therefore been considerable effort expended on increasing the amount of product which can be produced by a single eukaryotic host cell. Two of the main factors which control the amounts of product which a host cell can produce are gene copy number and the efficiency of transcription of each gene copy. There have been a number of proposals for increasing host cell productivity either by increasing gene copy number or by increasing transcription efficiency for each gene copy.
The most common method used to increase gene copy number is selection for gene amplification. In gene amplification, for instance as described in EP-A-0 045 809 or U.S. Pat. No. 4,634,665, a host cell is transformed with linked or unlinked genes. The first gene encodes a desired protein and the second gene encodes a selectable marker, such as DHFR. Cell lines containing both genes are then cultured in ever increasing concentrations of a toxic agent, the effect of which is nullified by the product of the selectable marker gene. It has been found that those cell lines which survive in the higher concentrations of the toxic agent have an increased copy number of both the selectable marker gene and the desired product gene. Thus, the host cell having the amplified number of gene copies can produce a larger amount of the desired protein than the original cell lines.
A disadvantage of gene amplification is that it is a laborious task to produce a highly productive cell line. Moreover, once the cell line has been produced, it is frequently necessary to retain it in a culture medium containing a high level of the selective agent. If this is not done, the selective pressure is released and the excess copies of the genes for the selectable marker and the desired protein may be eliminated. Using a culture medium containing a toxic agent causes problems in the purification of the desired product.
Another approach to increasing productivity is to increase the efficiency of transcription for each gene copy. This can be achieved by selection of a promoter which can be switched on by a stimulus, such as a toxic agent or heat. Alternatively, the gene for the desired product may merely be put under the control of a strong promoter. Although these approaches have to a certain extent been successful, they have not totally succeeded in raising productivity to commercially desirable levels.
There is therefore a need for a method for increasing the productivity of eukaryotic host cells containing genes encoding desired proteins.
It has been known for some long time that there are viral genes which, when translated, produce activator proteins which cause the activation of other genes in the viral genome. The activator proteins may act directly on recognition sites upstream of the viral promoter/enhancer sequence. Alternatively, the activator protein may interact with one or more other proteins which act on the recognition site to increase transcription. This activation of genes by an activator protein is often called transactivation.
It has also been found in a number of cases that an activating protein produced by one virus can act on recognition sites in other viruses or on certain cellular gene promoters to cause transactivation of the genes associated with the activation site.
References [1] to [30] listed in the attached bibliography are examples of the work done on transactivation, with particular reference to the Adenovirus E1A proteins.
The E1A region of adenoviruses encodes two major early proteins of 289 and 243 amino acids respectively. These are produced from two differently spliced mRNAs of 13S and 12S respectively. These E1A proteins are multifunctional and have been shown to play an essential role in cellular immortalization and transformation. The E1A gene product is also known to regulate the expression of certain viral and cellular genes in a positive or negative manner.
The 289 and 243 proteins are identical except in that the 243 protein lacks a 46 amino acid region towards the centre of the 289 protein. The 289 protein has been investigated and several functions have been assigned to certain domains thereof. The 289 protein is shown in FIG. 1 to which reference is now made. Domain 1 mediates induction of DNA synthesis. Domain 2 mediates induction of mitosis, cellular immortalisation and transformation, and transcription repression. Domain 3 mediates indirect transactivation of viral and cellular genes. Domain 4 mediates nuclear localisation.
The mechanism for gene repression and cellular transformation is unclear, but point mutations within Domain 2 are able to abolish both repression and transformation without affecting the other functions.
WO-A-89/05862 (Invitron) describes the use of E1A to immortalise or extend the life of cells from primary cultures which would otherwise senesce after a limited number of cell generations in culture. In particular, the application describes the use of E1A to extend the lifespan of the human colon mucosa cells CCD 18 Co to allow continuous secretion of the tPA produced natively from these cells. This approach exploits the known immortalisingxe2x80x94oncogene function of the 13S and 12S mRNAs from the E1A gene. It does not rely on transactivation.
More is known about the mechanism of transactivation, but even now the full mechanism has not been elucidated. It is known that the E1A 289 protein is phosphorylated and that somehow the active E1A protein induces phosphorylation of various DNA binding transcription factors, such as TFIId, ATF and TFIIIc, thus increasing their activity. It is conjectured that these activated transcription factors now bind to specific recognition sites, normally in homologous viral promoters, but also in mammalian genomic DNA sequences, to activate transcription from an associated promoter.
There have been various proposals for using transactivation to enable the production by recombinant DNA technology of a desired protein product. For instance, Grinnell et al. (see reference [19]) shows the production of a vector comprising the gene encoding human protein C (HPC) under the control of the adenovirus 2 late promoter in association with a BK virus (BKV) enhancer region. Another vector comprising the gene for the E1A 289 transactivator protein under the control of a BKV enhancer and promoter region is also used. The two vectors were used to cotransfect a human cell line. Cotransfection led to a 10 to 15 fold increase in HPC expression as compared to transfection with the first vector alone.
The system described by Grinnell [19] can be regarded as being essentially homologous, in that both BKV and adenoviruses are human viruses, the protein to be expressed is a human protein, and the host cell used for expression is a human cell. Despite the use of a homologous system, the level of expression achieved by Grinnell using the transactivator is relatively low.
U.S. Pat. No. 4,740,461 (Kaufman) generally relates to the use of amplifiable markers for increasing the yield of a desired protein. However, in Example 8, there is a description of the construction of a transcriptionally activated (transactivated) vector. The constructed vector contains an adenovirus 2 early region-2 (E2) promoter which is transcriptionally activated by the E1A gene transactivator protein. The adenovirus E2 promoter is used in separate vectors to control genes encoding DHFR and tPA. The E1A gene used is a fragment of total adenovirus. CHO cells are transfected with the two vectors and with the E1A gene. It is shown that the level of tPA production is decreased from 0.008 mU/cell/day to 0.0003 mU/cell/day if the E1A gene is omitted. However, even though an increased level of tPA production is achieved in the presence of E1A protein, the absolute level of tPA production is still relatively low. For instance, a CHO cell line transfected with a tPA gene under the control of the adenovirus major late promoter and SV40 enhancer can produce tPA at a level of 0.09 mU/cell/day without amplification or transactivation, and after selection for vector amplification can produce 6 mU/cell/day [35] or up to 10 mU/cell/day [U.S. Pat. No. 4,740,461].
WO88/07083 (Draper) generally relates to the use of transactivators for increasing the level of production of a desired protein. Although the disclosure is couched in general terms, and refers to such transactivators as ICP4, ICPO and ICP27 from herpes simplex virus (HSV), PRV-IE gene product from pseudorabies virus, E1A from adenovirus and HCMV-MIE protein from human cytomegalovirus, the main thrust of the disclosure relates to the use of the Vmw65 transactivator protein from HSV. It is to be noted that when referring to transactivators apart from the Vmw65 protein, the Draper application indicates that
xe2x80x9cthe structures, sequences or precise mechanism of action of the activation-reception sites for these stimulatory factors have not been fully elucidatedxe2x80x9d.
It is also to be noted that, although the Draper application shows that the use of the vmw65 protein can lead to enhanced levels of production, there are no absolute values given for the production level. It is therefore not clear whether the use of the Vmw65 protein system can give rise to a commercially viable expression system.
There are a number of reports of the insertion of adenovirus E1A genes into mammalian cultured cells to generate permanent E1A-expressing cell lines. For instance, Babiss et al. (reference [1]) transfected human BK cells with DNA containing and E1 region of adenovirus and could identify clones expressing E1A only, E1B only or both. Babiss et al. used the cloned cell lines to investigate the action of the adenovirus by carrying out complementation experiments.
Roberts et al. (reference [7]) used retroviral vectors to introduce DNA sequences encoding the 13S, 12S or 9S E1A mRNAa into mouse NIH-3T3 cells. Transfected cells expressing the 13S product could complement a mutant adenovirus defective in E1A function, thus demonstrating transactivation of E1B, E2, E3 and E4 promoters in the adenovirus. Effects on endogenous cellular gene expression were also noted. Some proteins were slightly induced while others, such as collagen and fibronectin, were repressed.
Bergman and Shavit (reference [29]) also transfected the E1A gene into NIH-3T3 cells and derived permanent E1A expressing cell lines. These cells were able to activate an immunoglobulin light chain (xcexa) promoter/enhancer in transient expression assays using a CAT reporter gene.
None of Babiss et al., Roberts et al and Bergman and Shavit in any way contemplated using the cloned cell lines for the commercial production of desired proteins by recombinant DNA technology.
Thus, there is an indication that the E1A gene can be introduced into cultured mammalian cells where its expression is compatible with continued cell proliferation and where it continues to function as a transactivator of transiently transfected or infected DNA. However, there is nothing in any of these reports which in any way suggests that transactivation could be useful in increasing production from genes inserted permanently into eukaryotic cell lines. Indeed, there are suggestions that this would not be feasible.
For instance, Brady et al [43] demonstrated that a transiently transfected rat insulin gene is activated in 293 cells although the chromosomal gene is not activated. The authors suggest that cellular insulin genes are in a chromosomal environment inaccessible to E1A or its associated transcription machinery. Alwine (reference [5]) provides evidence that, although transient expression from plasmids introduced into the adenovirus-transformed human 293 cell line is substantially higher than in cell lines not containing adenovirus sequences (see reference [3]), the principal cause of increased transient expression is greatly enhanced stability of extrachromosomal plasmid DNA. The increased stability is not solely due to E1A or E1B expression. Such an effect of increasing stability would not be observed in a permanently transfected cell line in which the DNA was integrated into the host genome.
Weisshaar (see reference [26]) used E1A in transient transfection experiments to reactivate an E2A promoter which was integrated into the genome of an adenovirus-transformed BHK cell line and which had become inactivated by methylation on initial integration. Reactivation was only partial if E1A was used alone. E1B was required in addition to E1A in order to achieve increased levels of expression from the E2A promoter. In a subsequent paper by the same group (Knxc3xcst et al.xe2x80x94reference [30]) it was shown that in adenovirus-transformed BHK cells, there is a constitutive level of endogenous E1A expression which, although detectable, is insufficient to reactivate the chromosomally integrated E2A gene.
The evidence of these papers therefore indicates that whereas E1A can activate transfected genes effectually in transient expression experiments, chromosomally located genes are only poorly activated, if at all. The levels of expression reported would not be commercially significant.
There is therefore a need for an expression system using transactivators which produces commercially viable quantities of a desired protein from a permanent cell line.
Accordingly, the present invention provides a method of producing a desired protein which comprises permanently transfecting a eukaryotic host cell with:
a first transcription unit containing a gene encoding a transactivator protein or a mutant thereof under the control of a promoter/enhancer region; and
a second transcription unit containing a gene encoding a desired protein under the control of a promoter/enhancer region which can be transactivated by the protein expressed by the first transcription unit,
wherein the promoter/enhancer region in the first transcription unit is selected so that the amount of transactivator protein expressed from the first transcription unit is not enough to prevent cell growth but is sufficient to transactivate the promoter/enhancer region in the second transcription unit so that the desired protein is expressed at a level at least twice that which would be obtained from the same cell but lacking the first transcription unit.
Preferably, the amount of desired protein expressed is at least five, the preferably ten, times that which would be obtained from the same cell but lacking the first transcription unit.
The promoter/enhancer region in each transcription unit is preferably a viral region, although exogenous eukaryotic promoter/enhancer regions may also be used.
Preferably, the gene in the first transcription unit encodes the E1A protein or a mutant thereof. However, any other viral transactivator protein, such as the Vmw65, ICP4, ICP0, ICP27, PRV-IE and HCMV-MIE proteins referred to above, mutants thereof, viral or other oncogene or proto-oncogene products such a myc, fos or jun, or mammalian cellular activators such as protein kinase A, protein kinase C or NFxcexaB, may be encoded by the first transcription unit.
In one alternative, the gene in the first transcription unit encodes the complete E1A 289 protein, in which case all four of its domains may be present. In another alternative, the first transcription unit encodes a mutant E1A 289 protein in which the amino acid sequence of the protein in one or more of domains 1, 2 and 4 has been altered in order to abolish or modify the activity of the or each domain. In a further alternative, the first transcription unit encodes a mutant E1A 289 protein which is substantially shorter than the native E1A protein.
Specific examples of mutant E1A 289 proteins which may be encoded by the gene in the first transcription unit are an E1A 289 protein in which mutations in Domain 2 have been made to abolish its repression function and an E1A 289-derived protein comprising the sequence of native Et1A protein from amino acid residues 139 to 289.
Preferably, the promoter in the first transcription unit is under the control of a weak promoter, such as the SV40E or SV40L promoter. Specific combinations which are preferred for the first transcription unit are the E1A gene under the control of the SV40E promoter and the R1176E1A gene (which is described below) under the control of the Sv40L promoter.
The promoter/enhancer region in the second transcription unit may be derived from any virus or cellular gene which includes a transactivator protein recognition site, the transactivator protein being one which is produced by or activated by the protein product of the gene in the first transcription unit. Such viruses include the adenoviruses (from one of which the E1A protein itself is derived) and the cytomegaloviruses (CMV), such as the human CMV (hCMV). In hCMV, the promoter/enhancer may be derived from the major immediate early gene. The selection of the promoter/enhancer region to be used in the second transcription unit will depend on the product of the gene in the first transcription unit and on the promoter/enhancer used in the first transcription unit.
Preferably, the promoter in the second transcription unit is a CMV-MIE promoter, especially when the gene in the first transcription unit encodes an E1A-derived protein.
The promoter/enhancer region in the first transcription unit may be any one of those referred to in the preceding paragraph, but will not necessarily be the same as or different from the promoter/enhancer region in the second transcription unit.
Particularly preferred combinations for use in the method of the present invention are, in the first transcription unit, either the E1A gene under the control of the SV40E promoter or the R1176E1A gene under the control of the SV40L promoter and, in the second transcription unit, the hCMV-MIE promoter controlling the gene for the desired protein.
The protein encoded by the gene in the second transcription unit may be, for instance, tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinases (TIMP), human growth hormone (hGH), insulin, and interferon, chymosin, calcitonin gene related polypeptide (CGRP) or an immunoglobulin heavy or light chain polypeptide, including chimeric, humanized and hybrid Ig polypeptides. It should be noted that this list is by no means exclusive and it will be readily apparent to the skilled person that any desired protein can be produced by the process of the present invention. The gene may encode only the protein sequence or may encode a pro- or prepro- protein.
It has been found by the present inventors that optimisation of the expression level of the activator is essential for obtaining a commercially useful level of expression of the desired protein. For instance, it has been found that, if native or mutant E1A gene products are expressed from a strong promoter, the transfection efficiency is reduced and the level of production from the surviving clones is not increased, which is contrary to expectations. It was envisaged that the more E1A protein produced, the more efficient would be transactivation, at least until a plateau of expression is reached. The results of transient expression experiments support this hypothesis since the stronger the promoter used to express the activator, the greater the transactivation in transient expression. However, in stable cell lines, relatively inefficient expression of the transactivator gene is required for optimal transactivation. In light of the findings of the present inventors, it is conjectured that overproduction of the activator is inhibitory to cell growth. Appropriate expression levels of activator can readily be obtained by choice of a suitable promoter/enhancer for the first transcription unit.
It is clear that it is not possible to use the results from transient expression systems to predict the effects of using the same transcription unit in stable cell lines. Thus, a skilled person investigating transactivation using transient expression systems would not have been led to the concepts underlying the present invention.
A further result which was not predicted is that a strong promoter can be used with a truncated version of the E1A 289 protein comprising only amino acids 139-289 of the native protein to achieve significant activation.
It will be appreciated that the observations made by the inventors and set out in this specification enable the skilled man to select the appropriate combination of promoter/enhancer regions and transactivator protein or mutant gene to enable a satisfactory level of desired protein to be produced.
It has surprisingly been found that transactivation can occur even if a strong promoter, such as the hCMV major immediate early promoter/enhancer, is used for the second transcription unit. It is generally considered that such promoters are already operating at optimal efficiency and that they could not be activated further. The present inventors have shown that this is not the case and have thus shown, surprisingly, that production from a single copy of a gene controlled by a strong promoter can be increased significantly, thus allowing commercially useful amounts of product to be obtained from low copy number vectors.
The eukaryotic host cell used may be a yeast cell, such as an S. Cerevisiae cell, but is preferably an animal cell such as an insect or mammalian cell. Suitable cells include CHO cells, L cells, HeLa cells and human or rodent lymphoid cells such as myeloma cells or hybridoma cells. Preferably the host cells are CHO or lymphoid cells.
The two transcription units may be present on a single vector, in which case the host cell will be transfected with the one vector to produce a suitably transfected host cell.
However, it is preferred that the two transcription units are present on separate vectors. In this case, the host cell may be simultaneously cotransfected with both vectors. Preferably, however, the host cell is transfected sequentially with the vectors. This will enable the production of a transactivated host cell line suitable for transfection by any one of a number of vectors encoding different desired proteins. Thus, the host cell will firstly be transfected with a vector containing the transactivator protein gene under control of a selected promoter/enhancer region. Thereafter, the cell can be transfected with a vector containing the second transcription unit wherein the gene for the desired protein is under the control of a promoter/enhancer region, for instance that of the hCMV-MIE gene, selected in accordance with the structure of the first transcription unit.
The or each vector containing the first and second transcription units may be, for instance, based on plasmids or on phages. Methods for producing such plasmid or phage vectors are well known in the art and are described, for instance, by Maniatis et al. [32].
In the transfected or cotransfected host cells, the first and second transcription units are integrated into the chromosomal material of the host cells.
In order to produce the protein, the transfected or cotransfected host cell will be cultured under appropriate conditions to cause the host cell to express the desired protein. Preferably, the host cell is selected such that the desired protein is correctly processed, for instance by glycosylation or removal of pro or prepro sequences, and secreted. Suitable methods for culturing transfected host cell lines are described by Bebbington and Hentschel [31].
According to another aspect of the present invention, there is provided a eukaryotic host cell which has been permanently transfected with a first transcription unit containing a gene encoding a transactivator protein or a mutant thereof under the control of a promoter/enhancer region which is selected so that the amount of transactivator protein expressed from the first transcription unit is not enough to prevent cell growth but is sufficient to transactivate the promoter/enhancer region in a selected second transcription unit so that a desired protein is expressible from said second transcription unit at a level at least twice that which would be obtained from the same cell containing said second transcription unit but lacking the first transcription unit.
The promoter/enhancer region in the first transcription unit is preferably of viral origin.
Preferably, the host cell is an animal, most preferably a mammalian cell such as a CHO or NSO cell. Where the host cell is derived from a CHO cell, it is preferred that its transcription unit should produce E1A or an E1A-derived transactivator protein. Most preferably, it should produce between 10 and 40%, particularly between 15 and 25%, for example about 20%, of the amount of mRNA encoding the transactivator protein as is produced by the 293 cell line (ATCC CRL 1573) referred to in reference, and further deposited on behalf of Lonza Group Ltd., on Jun. 19, 2002, with the ATCC, 10801 University Blvd, Manasas, Va. 20110-7209 (Telephone No. 703-365-2700; Facsimile No. 703-365-2745) and assigned Patent Deposit Designation PTA-4488.
The present invention also provides transactivated host cells, vectors for use in producing such host cells, and host cells transfected with both transcription units.